The linear mitochondrial genome of the

quarantine pest Synchytrium endobioticum;

Bart T. L. H. van de Vossenberg, Balázs Brankovics, Hai D. T. Nguyen,

Marga P. E. van Gent-Pelzer, Donna Smith, Kasia Dadej, Jarosław

Przetakiewicz, Jan F. Kreuze, Margriet Boerma, Gerard C. M. van

Leeuwen, C. André Lévesque and Theo A. J. van der Lee

Wart disease workshop 26-28 June 2019

Synchytrium endobioticum

▪ Soil-borne, obligate biotrophic (non-

culturable) fungus on potato causing wart

disease

▪ Wart formation on tubers and shoots

▪ Can result up to 100% yield loss

▪ Production of robust resting spores

(infectious > 40 years)

▪ World-wide quarantine status and on the

USA bioterrorism list

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Characterization of isolates

▪ As pathotypes based on their virulence on a reference set of potato

cultivars

▪ SSR markers

▪ TaqMan assay

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PJM Bonants, MPE van Gent-Pelzer… - European journal of plant pathology, 2015

MC Gagnon, TAJ van der Lee, PJM Bonants, DS Smith… - Phytopathology, 2016

Migration of S. endobioticum

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Can we gain insights into the evolution and recent history of introductions of this plant pathogen using the mitochondrial genome?

Why the mitochondrial genome?

Mitogenomes:

• do not mix or recombine with the

nuclear genome

• have a mutation rate about ten times

higher than nucDNA

• are relatively small, ranging up to

240 kb in fungi

• form a single haplotype/haplogroup

per organism

• have many copies (hundreds) per cell

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▪ One of the most ideal markers for monitoring the distribution and spread of populations is the mitochondrial genome (Harrison, 1989; Taylor, 1986).

▪ Mitochondrial genomes are relatively small and, therefore, can be studied in their entirety.

▪ Due to its high copy number within individual cells, the mitochondrial genome is easy to access.

▪ Simple organization that makes homologous regions easy to identify.

▪ Finally, in many fungal groups mitogenomes are inherited maternally (Basse, 2010),

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Why the mitochondrial genome?

https://doi.org/10.1016/0169-5347(89)90006-2https://doi.org/10.1016/0147-5975(86)90011-3https://doi.org/10.1016/j.mib.2010.09.003

▪ Mitochondrial sequences have been used for resolving phylogenetic and evolutionary relationships

between fungi at all taxonomic levels (Liu et al., 2009; Avila-Adame et al., 2006; Fourie et al., 2013).

▪ In 2003, the DNA barcoding initiative started, aiming at using a single marker for taxon identification.

The marker that was selected was a mitochondrial gene, cytochrome c oxidase I—COI or cox1 (Hebert

et al., 2003).

▪ The use of cox1 was abandoned as a barcoding region, because the frequent presence of introns in the

gene made this region impractical for PCR amplification (Gilmore et al., 2009).

▪ Next generation sequencing (NGS) and new analysis methods have resolved this issue by dispensing

with the need for PCR amplification for extracting mitochondrial sequences (Brankovics et al., 2016).

▪ In addition, de novo assembly of mitochondrial sequences from NGS data is not confounded by the

presence of nuclear mitochondrial DNA segments (NUMTs), while NUMTs are known to cause problems in

PCR-based barcoding (Song et al., 2008).

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Why the mitochondrial genome?

https://doi.org/10.1186/1471-2148-9-272https://doi.org/10.1007/s00294-005-0016-3https://doi.org/10.1186/1471-2164-14-605https://doi.org/10.1098/rspb.2002.2218https://doi.org/10.1111/j.1755-0998.2009.02636.xhttps://doi.org/10.1371/journal.pcbi.1004753https://doi.org/10.1073/pnas.0803076105

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“Next generation sequencing (NGS) and new analysis methods have resolved this issue by dispensing with the need for PCR amplification for extracting mitochondrial sequences”

Assembly of the mitochondrial genome

▪ Individual assemblies were aligned to create a consensus mtDNA assembly

▪ The consensus mtDNA assembly was annotated using the online Mfannot tool

Mfannot: University of Montreal; http://megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannotInterface.pl.

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Assembly of the mitochondrial genome

http://megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannotInterface.pl

The consensus mtDNA

▪ Assembly size: 72.8 kb

▪ No circular confirmation found

▪ Drop in read coverage at 5’ and 3’ ends: consistent with linear

mtDNA hypothesis

▪ Inverted repeats at 5’ and 3’ ends (~3 kb)

▪ All 14 “core” genes for fungi, 5 tRNAs and 2 rRNAs predicted

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Primer design for assembly verification

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Improved procedures

▪ New DNA extraction protocol shows high molecular DNA extract

▪ WGA step to generate sufficient material for confirmation

experiments

▪ Fragmentation controls show amplification up to 4.3 kb

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Verification of linearity of the S. endobioticum mtDNA

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Assembly and Annotation of the linear

mtDNA▪ Three assemblies were

used to determine mtDNA

genome sequence

▪ “standard” fungal mtDNA

genes identified

▪ Reduced set of tRNAs

▪ GC-rich intergenic regions

(up to 68.5%)

▪ Codes for dpoB and intron

encoded endonucleases

▪ Linear with terminal

inverted repeats (TdT

tailing and sanger

sequence verified) 14

Linear mtDNA genomes in Chytrid species

▪ Bayesian Inference of

phylogeny with high support

for all nodes

▪ Three linear mtDNA genomes

are known in Chytrid

species: Sendo, Bdend, and

Hcurv

● Independent events

▪ Splits the Chytridiales

▪ Little intraspecies variation

for Sendo (19

polymorphisms)15

Linearization of the S. endobioticum

mitogenome is a recent event

▪ S. microbalum: conserved organization and orientation; circular

mapping, no endonucleases or dpoB

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mtDNA haplotypes reveal 4 major groups

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• Build on 141 informative sites (SNPs) from the entire mtDNA

More than one haplotype per sample

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▪ mtDNA haplotype was build on 141

informative sites

▪ SNPs do not show a black and white

distribution

● Intermediate SNP frequencies

● >1 mtDNA haplotype is present in many samples

▪ SNP frequency distribution and haplotype

composition seems to be fairly conserved

for mtDNA groups

mtDNA haplotype diversity

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Conclusions

▪ The S. endobioticum mtDNA genome is linear and shows that the

pest has been introduced in Europe at least three times

▪ Several pathotypes emerged more than once independently

▪ S. endobioticum isolates are populations and have more than one

haplotype per sample

▪ These findings have an impact on breeding for potato wart

resistance, as the diversity of S. endobioticum virulence is

underestimated

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Acknowledgements

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Biointeractions and Plant Health

Bart van de Vossenberg, Marga van Gent-

Pelzer, Balazs Brankovics, Peter Bonants,

Theo van der Lee

Bioinformatics

Sven Warris, Henri van de Geest, Linda

Bakker

Plant Breeding

Jack Vossen, Gert van Arkel, Marjan

Bergervoet, Charlotte Prodhomme

Promotor Richard Visser

Other

▪ Margriet Boerma (HLB, NL)

▪ Bart van de Vossenberg, Gerard van

Leeuwen, Patricia van Rijswick, Annebeth

Kloosterman, Nico Mentink (NVWA, NL)

▪ Hai Nguyen, Kasia Dadej (AAFC, Canada)

▪ André Levesque, Donna Smith (CFIA,

Canada)

▪ Jarek Przetakiewicz (IHAR, Poland)

▪ Jan Kreuze (CIP, Peru)

Funding TKI wart project Averis seeds BV,

Boehm-Nordkartoffel Agrarproduktion GmbH &

Co. OHG, HZPC Holland, SaKa Pflanzenzucht

GmbH & Co. KG, Taegasc, Meijer Potato, LKF

Vandel, HLB BV and NVWA